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Values are in % product formation/min/nmol enzyme relative to incubations with BM3 R47L/F87V/L188Q. For amodiaquine, N-desethylamodiaquine was compared with N-desethylamodiaquine formed by P450 1A1, P450 2C8, and P450 2D6. For dextromethorphan, 3-methoxymorphinan was compared with 3-methoxymorphinan formed by P450 2D6. For testosterone, the major metabolite was compared with 6β-hydroxytestosterone formed by P450 3A4. For MDMA, 3,4-OH-MA was compared with 3,4-OH-MA formed by P450 2D6. For APAP, NAPQI was compared with NAPQI formed by P450 3A4.Full-size tableTable optionsView in workspaceDownload as CSVDextromethorphan was N-demethylated by the BM3 triple mutant to form 3-methoxymorphinan (Fig. 2B). This metabolite was identified by LC–MS as described previously (m/z 258; MS/MS m/z 215) [24]. The specific activity of dextromethorphan N-demethylation by the BM3 triple mutant was 12.5-fold lower than that of P450 2D6 (Table 1).Testosterone was metabolised by BM3 R47L/F87V/L188Q to three metabolites (Fig. 2C). The major metabolite had a tr of 10.0 min, the second highest metabolite had a tr of 16.1 min and the third metabolite had a tr of 19.1 min. All three metabolites were identified by LC–MS as monohydroxylated metabolites (m/z 305). In addition, trace amounts of a dihydroxylated metabolite (m/z 321) at tr 2.6 min were observed with MS. The metabolite at tr 16.1 min co-eluted with a reference standard of 16β-hydroxytestosterone. Of the double and single mutants, only the two double mutants F87V/L188Q and R47L/F87V showed metabolism of testosterone. The same metabolites were formed as by the triple mutant, but the amounts were 4- and 10-fold lower, respectively (data not shown). P450 3A4 is known to form mainly 6β-hydroxytestosterone. Comparison of incubations with P450 3A4 and BM3 R47L/F87V/L188Q showed that the BM3 triple mutant does not form 6β-hydroxytestosterone. The specific activity of the formation of the metabolite tr 10.0 by BM3 R47L/F87V/L188Q was approximately 2-fold higher than the specific activity of testosterone 6β-hydroxylation by P450 3A4, assuming that the Granisetron Hydrochloride coefficient of both metabolites is similar (Table 1).MDMA was metabolised by BM3 R47L/F87V/L188Q to three metabolites (Fig. 2D). The major metabolite had a tr of 8.8 min and was identified as MDA by coinjection with a reference standard. The second metabolite, with tr 17.2 min, was identified as N-OH-MDMA by coinjection with a reference standard [24]. The minor metabolite with tr 3.8 min was identified previously as 3,4-dihydroxy-methylamphetamine (3,4-OH-MA) using electrochemical detection [24]. Comparison with an incubation with P450 2D6 showed that the specific activity of 3,4-OH-MA formation by BM3 R47L/F87V/L188Q was 35-fold lower than that of P450 2D6 (Table 1). Of the double and single mutants, only the double mutant F87V/L188Q was able to metabolise MDMA. The amount of metabolite formed was so low that only MDA was detectable (data not shown).APAP was metabolised by BM3 R47L/F87V/L188Q to one metabolite with a tr of 17.5 min (Fig. 3). This peak was identified as the NAPQI-SG conjugate by coinjection with a reference standard synthesised as described [28] and [29]. The specific activity of NAPQI formation by BM3 R47L/F87V/L188Q was approximately 15-fold lower than that of P450 3A4 (Table 1).Fig. 3. HPLC chromatograms of BM3 wild-type and BM3 R47L/F87V/L188Q incubations with APAP. Black line: wild-type BM3. Dark grey line: BM3 R47L/F87V/L188Q. Light grey line: BM3 R47L/F87V/L188Q with 5 mM caffeine. The NAPQI-SG peak is indicated in the chromatogram.Figure optionsView in workspaceDownload full-size imageDownload as PowerPoint slideDetermination of kinetic parametersFor MDMA metabolism, the 3,4-OH-MA peak was too small to determine accurate kinetic parameters. No saturated V/[S] curve could be obtained for MDA- and N-OH-MDMA formation with the substrate concentration range used for this experiment (Fig. 4). Therefore, the slope of the linear part of the V/[S] plot was used to determine the catalytic efficiency Vmax/Km (Table 2). Compared to MDA formation by P450 2D6[F120A], the catalytic efficiency of BM3 R47L/F87V/L188Q was approximately 700-fold lower [30].Fig. 4. V/[S] curves of MDMA, APAP, and testosterone metabolism by BM3 R47L/F87V/L188Q. For MDA formation from MDMA (left) and NAPQI formation from APAP (middle), V/[S] curves obtained in the absence (squares) and presence (triangles) of caffeine are fitted with the Michaelis–Menten equation. A zoom-in of the APAP graph is displayed in the inset. For testosterone metabolism (right), the V/[S] curve of the major metabolite tr 10.0 is fitted with the Hill equation. In the inset, a Hill plot is displayed (Log[V/(Vmax ? V)] vs Log[S]).Figure optionsView in workspaceDownload full-size imageDownload as PowerPoint slideTable 2. Kinetic parameters of MDMA and APAP metabolism by BM3 R47L/F87V/L188Q in the presence and absence of caffeineSubstrate (product)Without caffeine